Reduction of Int6 induces CAF-like properties in normal human mammary fibroblasts. (A) HMFs were transfected with control or anti-INT6 siRNA. On the left, protein samples were analyzed over time by Western blots using antibodies against α-SMA and Int6. GAPDH was the loading control. On the right, a quantification of Western blots from three separate experiments was graphed. (B) HMFs were treated with control or anti-INT6 siRNA for 5 days and then immunostained with antibodies against α-SMA (red) and tubulins (green), and counter-stained by DAPI (blue) to mark the nuclei. Cells containing α-SMA cables were counted (n = 100 cells). White arrow indicates a typical α-SMA cable in a cell. Quantification of cells containing α-SMA cables is shown on the right. (C) On the left, semiquantitative RT-PCR was performed to measure CXCL12 (encoding SDF-1) mRNA levels in parental and Int6-repressed HMFs. CXCL12 mRNA levels were normalized to those of ACTB, and the normalized CXCL12 mRNA levels in the control were set to 1. (D) The growth media of the cells from panel C were analyzed by ELISA to measure SDF-1 levels 7 days after seeding. SDF-1 levels in the control were set to 1 (n = 3 separate experiments).